REGULATION OF TNF-ALPHA-INDUCED REORGANIZATION OF THE ACTIN CYTOSKELETON AND CELL-CELL JUNCTIONS BY RHO, RAC, AND CDC42 IN HUMAN ENDOTHELIAL-CELLS

We have investigated the role of the small guanosine-trisphosphate (GT P)-binding proteins, Rho, Rac, and Cdc42, in the early responses of hu man umbilical vein endothelial cells (HUVECs) to TNF-alpha (tumor necr osis factor-alpha). Quiescent confluent HUVECs incubated with TNF-alph a for 5-30 min showed an increased formation of membrane ruffles, filo podia, and actin stress fibres followed by cell retraction and formati on of intercellular gaps. This process was accompanied by the dispersi on of cadherin-5 from intercellular junctions. TNF-alpha also induced a transient increase in polymerized F-actin, as determined both by mea suring C-actin content and by quantifying fluorescent emission from fl uorescein isothiocyanate (FITC)-phalloidin-labelled F-actin. Microinje ction of cells with activated RhoA protein led to an increase in polym erized actin, formation of stress fibres, cell retraction as well as d ispersion of cadherin-5. The proteins Cdc42 and Rac induced qualitativ ely similar effects to Rho, although not as dramatic and in addition i nduced formation of filopodia and lamellipodia. Microinjection of cell s with a Rho inhibitor, C3 transferase, prevented gap formation caused by TNF-alpha. Similar effects were observed in cells microinjected wi th the dominant inhibitory proteins N17Cdc42 and N17Rac1. Cell retract ion and gap formation were also prevented by inhibitors of myosin ligh t chain kinase (MLCK). Our data suggest that Cdc42, Rac, and Rho are a ctivated in a hierarchical cascade following stimulation with TNF-alph a leading to actomyosin-mediated cell retraction and formation of inte rcellular gaps, (C) 1998 Wiley-Liss, Inc.